Peptides, compositions and uses thereof

ABSTRACT

Described is an N-terminal hexapeptide fragment of maximakinin, QUB 698.8, which exhibits potent tissue selective actions on smooth muscle. It demonstrates a high degree of selectivity for arterial smooth muscle over small intestinal smooth muscle, causing potent relaxation of arterial smooth muscle, while causing less potent contraction of ileal smooth muscle. It may be used treatment of diseases of the cardiovascular system and in promotion of angiogenesis.

FIELD OF THE INVENTION

The present invention relates to peptides, compositions and usesthereof. In particular it relates to peptides, which are based on theN-terminal hexapeptide of maximakinin (syn. bombinakinin M) and whichhave cell growth stimulatory activity, particularly in promotingangiogenesis, and uses thereof in methods of therapy.

INTRODUCTION

The defensive secretions from the dermal granular or poison glands ofamphibians, particularly those of anurans, are complex molecularcocktails containing proteins, biogenic amines, alkaloids and a plethoraof bioactive peptides (Lazarus, L. H. and Atilla, M. (1993) Prog.Neurobiol. 41, 473-507). Many skin peptides exhibit high degrees ofstructural similarity with endogenous vertebrate regulatory peptides butare usually more bioactive as a consequence of structural modificationsoccurring outside the conserved bioactive core sequence, an attributeproduced by their natural selection for an exogenous delivery mode(Erspamer et al (1985) Peptides 6, Suppl. 3, 7-12). Caerulein, bombesin,dermorphin and deltorphin are examples of amphibian skin peptides thatdisplay enhanced activity at endogenous vertebrate cholecystokinin(CCK), gastrin-releasing peptide (GRP) and μ- and δ opioid receptors,respectively (Anastasi et al (1971) Experientia 27, 166-167; Anastasi etal (1968) Archs. Biochem. Biophys. 125, 57-68; Broccardo et al (1981)Br. J. Pharmacol. 73, 625-631; Kreil et al (1989) Eur. J. Pharmacol.162, 123-128 10).

A number of bradykinin-like peptides have been isolated from amphibianskin secretions, some of which are believed to be associated withdefence mechanisms of the Amphibia (Conlon and Aronsson (1997) Peptides18, 361-365; Yasuhara et al (1979) Chem. Pharm. Bull. (Tokyo) 27,486-491; Nakajima, T. (1968) Chem. Pharm. Bull. (Tokyo) 16, 769-774;Anastasi et al (1966) Br. J. Pharmacol. 27, 479-485; Yasuhara et al(1973) Chem. Pharm. Bull. (Tokyo) 21, 138-139). Lai et al (2001)Biochem. Biophys. Res. Commun. 286, 259-263 described the isolation of abradykinin related peptide from the skin secretions of the toad Bombinamaxima. Although this peptide, named bombinakinin M, was found to havecontractile activity on guinea pig ileum smooth muscle, the authors ofthis paper suggest that the physiological role of the peptide isunclear.

In WO 2004/069857, which shares the principal inventor with the presentapplication, an N-terminally extended bradykinin, maximakinin, isdescribed. Maximakinin has amino acid sequence:Asp-Leu-Pro-Lys-Ile-Asn-Arg-Lys-Gly-Pro-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg(SEQ ID NO: 1). Maximakinin, which has the same amino acid sequence asbombinakinin M (Lai et al (2001) Biochem. Biophys. Res. Commun. 286,259-263) was found to demonstrate tissue selectivity in its actions onsmooth muscle. Also identified in WO 2004/069857 were a number ofC-terminal active fragments of maximakinin.

SUMMARY OF THE INVENTION

The present inventors have studied N-terminal fragments of maximakininand have surprisingly demonstrated that, contrary to expectations, anN-terminal fragment of maximakinin, exhibits potent tissue selectiveactions on smooth muscle. Specifically, it was found that, despite theabsence of the C-terminal residues of maximakinin, which were previouslythought to be necessary for its activity, an N-terminal hexapeptideherein referred to as QUB 698.8, demonstrates a high degree ofselectivity for arterial smooth muscle over small intestinal smoothmuscle, causing potent relaxation of arterial smooth muscle, whilecausing less potent contraction of ileal smooth muscle.

Moreover, not only does such an N-terminal fragment retain the tissueselective properties of maximakinin, but it is significantly more potentthan maximakinin.

Furthermore, the inventors unexpectedly found that, despite the previousstudies involving maximakinin suggesting that the pro-angiogenic effectsof this molecule and fragments thereof were also dependent on thepresence of C-terminal residues of maximakinin, the N-terminalhexapeptide QUB698.8 demonstrates potent pro-angiogenic activity.

Accordingly, in a first aspect, the present invention provides apro-angiogenic peptide, wherein said pro-angiogenic peptide is QUB698.8or a biologically active fragment or derivative thereof.

QUB698.8 has the amino acid sequence:

Asp-Leu-Pro-Lys-Ile-Asn (SEQ ID NO: 2)

In one embodiment the pro-angiogenic peptide of the invention consistsof the amino acid sequence

Asp-Leu-Pro-Lys-Ile-Asn (SEQ ID NO: 2)

In the context of the present invention, a fragment of QUB698.8 is apeptide having at least five contiguous amino acids corresponding to asegment of QUB698.8 having the same five contiguous amino acids.

Derivatives of QUB698.8 include peptides having the amino acid sequenceof SEQ ID NO: 2 with one, two, or three amino acid substitutions,insertions, deletions, or a combination thereof.

In one embodiment, the derivative comprises at least five contiguousamino acids of QUB 698.8.

Specific derivatives include:

Leu-Pro-Lys-Ile-Asn (SEQ ID NO: 3) Asp-Leu-Pro-Lys-Ile (SEQ ID NO: 4)Asp-Leu-Pro-Lys-Ile-Asn-Arg-Lys (SEQ ID NO: 5)Leu-Pro-Lys-Ile-Asn-Arg-Lys-Gly (SEQ ID NO: 6)Asp-Leu-Pro-Lys-Ile-Asn-Arg-Lys-Gly (SEQ ID NO: 7)Leu-Pro-Lys-Ile-Asn-Arg-Lys-Gly-Pro. (SEQ ID NO: 8)

Furthermore, derivatives may comprise additional amino acids at the N-and/or C-terminal. In such embodiments, the derivative preferablycomprises one to eleven additional amino acids for example, two, three,four, five, six, seven, eight, nine, ten or eleven amino acids. In thecontext of the present application, such derivatives do not include apeptide having the amino acid sequence of maximakinin itself.

Such fragments and derivatives preferably retain pro-angiogenicactivity.

Unless the context demands otherwise, reference to peptides of theinvention encompasses such derivatives and fragments.

According to a second aspect of the present invention there is provideda polynucleotide encoding a peptide according to the first aspect of theinvention, wherein said polynucleotide does not encode a polypeptidecomprising an amino acid sequence shown as Sequence ID No: 1.

According to a third aspect of the present invention there is provided amethod of inducing relaxation of arterial smooth muscle cells or tissue,said method comprising administering a pro-angiogenic peptide of thefirst aspect of the invention, or a polynucleotide according to thesecond aspect of the invention to said cell or tissue.

In one embodiment of the invention, the EC50 of the pro-angiogenicpeptide of or for use in the invention at arterial smooth muscle is lessthan the EC50 of maximakinin at arterial smooth muscle cells, and/or theEC50 of the pro-angiogenic peptide of the invention at small intestinalcells is more than the EC50 of maximakinin at small intestinal cells.

The surprising demonstration that the peptides of the invention exhibitsuch potent, yet selective effects on smooth muscle receptors, enablesthe use of such peptides in the treatment of diseases of thecardiovascular system while minimising side effects associated with theuse of less selective agents.

Accordingly, in a fifth aspect of the present invention, there isprovided a method of promoting vasodilation in a tissue, comprisingadministering a peptide according to the first aspect of the inventionor a polynucleotide according to the second aspect of the invention, tosaid tissue.

The peptides, polynucleotides and methods of the invention may beparticularly useful in the treatment of cardiovascular disease whileminimising unwanted side effects, for example on the gastrointestinalsystem.

Accordingly, in a sixth aspect of the invention, there is provided amethod of treating a cardiovascular disease, said method comprisingadministering a therapeutically effective amount of a peptide of thefirst aspect of the invention or a polynucleotide according to thesecond aspect of the invention to a patient in need thereof.

Cardiovascular diseases for which the present invention may be usedinclude any cardiovascular disease, condition or symptom for whichvasodilation may be useful. For example, in one embodiment, thecardiovascular disease is hypertension. In another embodiment, thecardiovascular disease is pulmonary hypertension. In a furtherembodiment, the cardiovascular disease is coronary artery disease. In afurther embodiment, the cardiovascular disease is peripheral vasculardisease. In a further embodiment, the cardiovascular disease is anischemic condition, for example angina or and stroke. Other diseases forwhich the peptides and methods of the invention may be used include thetreatment of vasospasm, for example, intra- or post-operatively, anderectile dysfunction.

The invention may be used to treat acute conditions where acutevasodilation may be beneficial.

As described above, the inventors have also surprisingly shown that thepeptides of the invention demonstrate potent pro-angiogenic effects.Thus the data disclosed herewith is indicative of a potent role for thepeptides of the invention in promoting the growth of new blood vessels.

Thus according to a seventh aspect of the present invention there isprovided the use of a pro-angiogenic peptide according to the firstaspect of the invention or a polynucleotide according to the secondaspect of the invention in promoting angiogenesis in a biologicalsample.

In the context of the present invention, references to promotingangiogenesis should be understood to refer to inducing or acceleratingan angiogenic taxic response or the inducing or accelerating ofangiogenesis per se i.e. vessel formation.

According to an eighth aspect of the present invention there is provideda method of stimulating angiogenic taxic responses in cells comprisingadministering a pro-angiogenic peptide according to the first aspect ofthe invention or a polynucleotide according to the second aspect of theinvention to said cells.

An angiogenic taxic response in cultured endothelial cells involvesproduction of sites of attachment between cells mediated by club-likeprojections and orientation of cells into pre-angiogenic tubes. It maybe assessed visually using a microscope.

The methods of the invention may be practised in vitro or in vivo. Wherepractised in vivo, the methods of the invention may be used in methodsof treatment of animals or humans.

According to a ninth aspect of the present invention there is provided amethod of promoting angiogenesis comprising the step of administering apeptide according to the first aspect of the invention or apolynucleotide according to the second aspect of the invention at atherapeutically effective amount to a patient in need thereof.

The promotion of angiogenesis may be useful in a number of clinicalconditions. For example, the pro-angiogenic peptide(s) of the inventionmay be used to promote angiogenesis of collateral vasculature inmyocardial tissue during or following ischaemic disease, myocardialinfarction or following coronary bypass surgery. Other diseases orconditions which may be treated by the provision of the peptide(s) ofthe invention include vascular disease and/or ischaemic disease causingpathology of the peripheral or central nervous system. Suchconditions/diseases may include cerebrovascular accidents, e.g. causedby clot occlusions or by rupture of aneurysms, or general/localisedischaemia causing neuronal death or peripheral functional impairmentsuch as in motor or sensory functions or speech impairment. Thus, in oneembodiment, the peptides, methods and uses of the invention are for thetreatment of cerebrovascular accidents. In another embodiment, thepeptides, methods and uses of the invention are for use in the treatmentof ischaemic disease, which may be general or localised.

Furthermore, the pro-angiogenic properties of the peptide(s) of theinvention may be used in the promotion of tissue repair, for example,wound healing. Accordingly, in a seventh aspect of the invention thereis provided the use of a peptide of the invention in promoting tissuerepair and/or the treatment of a wound.

According to a tenth aspect of the present invention there is provided amethod of promoting tissue repair, said method comprising the step ofadministering a peptide according to the first aspect of the inventionor a polynucleotide according to the second aspect of the invention at atherapeutically effective amount to a patient in need thereof.

The peptide(s) and methods of the invention may be used in the repair ofany suitable tissue in need thereof. For example, the peptide(s) may beused in the repair of any damaged tissue or the healing of any woundwhere vascular angiogenesis and/or revascularisation is desired. Forexample, in one embodiment, wounds which the peptide(s) and methods ofthe invention may be used to treat include ulcers, such as dermalulcers, for example pressure sores, venous ulcers, and diabetic ulcers.In another embodiment, the methods and peptide(s) may be used to treattissue trauma. In one embodiment, the tissue trauma is a wound such as alaceration. In another embodiment, the tissue trauma which the presentinvention may be used to treat is a burn. The peptides and methods maybe particularly useful in applications where transplanted tissues, forexample, skin grafts, are employed in the repair of such wounds.

In vascular graft surgery, the peptides and methods of the invention maybe used to promote endothelialization. Where vascular grafts usingeither transplanted or synthetic material are used the peptides may beapplied to the surface of the graft to promote the growth of vascularsmooth muscle and adventitial cells in conjunction with endothelialcells. Furthermore, where materials, natural or synthetic, are to betransplanted into the body, the peptides of the invention may be used tocoat the materials to reduce and/or minimize rejection of thetransplanted material and to stimulate vascularization of thetransplanted materials.

Thus in an eleventh aspect, the invention provides a method of treatingor preventing transplant rejection in a subject, said method comprisingthe step of administration of a peptide according to the first aspect ofthe invention or a polynucleotide according to the second aspect of theinvention to said subject.

Such peptides may also be useful in the repair of vascular damage causedby arteriosclerosis or damage caused by balloon angioplasty. Bypromoting repair of such tissues, damage may be minimised andre-stenosis may be prevented. Thus in a further embodiment of theinvention, there is provided a method of treating vascular damage in asubject, said method comprising the step of administration of a peptideaccording to the first aspect of the invention or a polynucleotideaccording to the second aspect of the invention to said subject

Further, more generally, the peptides of the invention also exhibitpotency in effecting cell growth; for example of cells located on thesurface of a body or internally. In particular the peptide of theinvention eg. QUB 698.8, may be used to promote the growth of dermalcells.

Thus, the invention may also be used to treat conditions such as burns.This property of the peptides of the invention may also be used in thepromotion of graft revascularisation and in the promotion of healingpost-operatively, for example after cosmetic surgery.

According to a twelfth aspect of the invention, there is provided apharmaceutical composition comprising a peptide according to the firstaspect of the invention or a polynucleotide according to the secondaspect of the invention, and a pharmaceutically acceptable carrier orexcipient.

Furthermore, in a thirteenth aspect, there is provided a peptideaccording to the first aspect of the invention or a polynucleotideaccording to the second aspect of the invention for use in medicine.

A fourteenth aspect of the invention provides a peptide according to thefirst aspect of the invention or a polynucleotide according to thesecond aspect of the invention for use in the treatment of acardiovascular disease.

A fifteenth aspect of the invention provides a peptide according to thefirst aspect of the invention or a polynucleotide according to thesecond aspect of the invention for use in the treatment of a wound or intissue repair.

A sixteenth aspect of the invention provides a peptide according to thefirst aspect of the invention or a polynucleotide according to thesecond aspect of the invention for use in preventing or inhibiting graftor transplant rejection.

A seventeenth aspect of the invention provides according to the firstaspect of the invention or a polynucleotide according to the secondaspect of the invention in the preparation of a medicament for thetreatment of cardiovascular disease.

An eighteenth aspect of the invention provides the use of a peptideaccording to the first aspect of the invention or a polynucleotideaccording to the second aspect of the invention in the preparation of amedicament for the treatment of a wound or for tissue repair.

A nineteenth aspect of the invention provides the use of a peptideaccording to the first aspect of the invention or a polynucleotideaccording to the second aspect of the invention in the preparation of amedicament for the treatment of graft or transplant rejection.

A twentieth aspect of the present invention is a wound dressingcomprising a peptide of the first aspect of the invention or apolynucleotide according to the second aspect of the invention.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis.

DETAILED DESCRIPTION

Peptides of and for use in the present invention include QUB 698.8 (SEQID NO: 2), derivatives and fragments thereof, in particular thederivatives and fragments described above. Herein, the term derivativeis used interchangeably with analogue.

In one embodiment, a “fragment” or derivative of QUB 698.8 or for use inthe present invention retains the ability of QUB 698.8 to relax arterialsmooth muscle with an EC50 less than the EC50 of its constrictory effecton ileal muscle.

In another embodiment, the fragment or derivative of QUB 698.8 retainsthe ability to promote angiogenesis. Such ability may be assessed usingany known technique, for example using an endothelial cell angiogenictubule formulation assay, an endothelial cell wound scrape essay or anMTT assay, as for example as described in the Examples.

In one embodiment the fragments or derivatives of the invention retainboth the arterial smooth muscle dilatory ability and pro-angiogenicability as defined above.

Other derivatives of the QUB 698.8 peptides of and for use in theinvention include multimeric or fusion peptides including QUB 698.8peptides, analogue or fragments of the invention, and prodrugs includingsuch sequences, the peptide linked to a coupling partner, e.g. aneffector molecule, a label, a drug, a toxin and/or a carrier ortransport molecule. Techniques for coupling the peptides of theinvention to both peptidyl and non-peptidyl coupling partners are wellknown in the art.

Derivatives of and for use in the present invention further includereverse- or retro-analogues of QUB 698.8 peptides or their syntheticderivatives. See, for example, EP 0497 366, U.S. Pat. No. 5,519,115, andMerrifield et al., 1995, PNAS, 92:3449-53, the disclosures of which areherein incorporated by reference. As described in EP 0497 366, reversepeptides are produced by reversing the amino acid sequence of anaturally occurring or synthetic peptide. Such reverse-peptides retainthe same general three-dimensional structure (e.g., alpha-helix) as theparent peptide except for the conformation around internalprotease-sensitive sites and the characteristics of the N- andC-termini. Reverse peptides are purported not only to retain thebiological activity of the non-reversed “normal” peptide but may possessenhanced properties, including increased biological activity. (SeeIwahori et al., 1997, Biol. Pharm. Bull. 20: 267-70). Derivatives of andfor use in the present invention may therefore comprise reverse peptidesof natural and synthetic QUB 698.8 peptides.

Indeed, such reverse QUB 698.8 peptides and derivatives and fragmentsthereof constitute a further independent aspect of the presentinvention. Preferably such reverse QUB 698.8 peptides retain biologicalactivity such as the ability to induce pro-angiogenic responses withincells and tissues and/or the ability to selectively relax arterialsmooth muscle.

In one embodiment, a reverse peptide of the invention has the reverseamino acid sequence of the peptide shown in SEQ ID NO. 2.

Thus, in one embodiment of the invention, the reverse peptide has theamino acid sequence:

Asn-Ile-Lys-Pro-Leu-Asp (SEQ ID NO: 9)

Peptides (including derivatives, reverse peptides and fragments ofeither), of and for use in the invention, may be generated wholly orpartly by chemical synthesis or by expression from nucleic acid. Forexample, the peptides of and for use in the present invention can bereadily prepared according to well-established, standard liquid or,preferably, solid-phase peptide synthesis methods known in the art (see,for example, J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984),in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis,Springer Verlag, New York (1984)).

Multimeric Peptides

As noted above, peptides of the invention may be in the form ofmultimers. Thus multimers (for example of 2, 3 or more individual QUB698.8 analogue monomeric units or fragments) are within the scope of theinvention.

Such multimers may be used to prepare a monomeric peptide by preparing amultimeric peptide that includes the monomeric unit, and a cleavablesite (i.e., an enzymatically cleavable site), and then cleaving themultimer to yield a desired monomer.

The use of multimers can increase the binding affinity for a receptor.Thus, in the present case, the binding affinity of the peptides of theinvention to their receptors, could be increased by using multimers of2-5, for example 2-3 receptor binding moieties.

The multimers can be homomers or heteromers. As used herein, the termhomomer, refers to a multimer containing only polypeptides correspondingto the amino acid sequence of SEQ ID NO. 2 or fragments thereof, orother QUB 698.8 derivatives described herein. These homomers may containpro-angiogenic peptides of the invention having identical or differentamino acid sequences. For example, the multimers can include onlypro-angiogenic peptides of the invention having an identical amino acidsequence, or can include fragments or derivatives of pro-angiogenicpeptides. The multimer can be a homodimer (e.g., containingpro-angiogenic peptides having identical or different amino acidsequences), homotrimer or homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., peptides which are not QUB698.8 peptides, fragments or derivatives thereof) in addition to QUB698.8 peptides, fragments or derivatives thereof described herein.

The multimers may be the result of hydrophobic, hydrophilic, ionicand/or covalent associations and/or may be indirectly linked by, forexample, liposome formation. Thus, in one embodiment, multimers may beformed when the pro-angiogenic peptides of the invention describedherein contact one another in solution. In another embodiment,heteromultimers may be formed when pro-angiogenic peptides of theinvention and peptides which are not QUB 698.8 peptides, fragments orderivatives thereof contact antibodies to the polypeptides describedherein (including antibodies to the heterologous polypeptide sequence ina fusion protein described herein) in solution. In other embodiments,multimers described herein may be formed by covalent associations withand/or between the pro-angiogenic peptides of the invention (andoptionally peptides which are not QUB 698.8 peptides, fragments orderivatives thereof) described herein.

Such covalent associations can involve one or more amino acid residuescontained in the QUB 698.8 sequence (e.g., that recited in SEQ ID NO.2). In one embodiment, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations can involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a QUB 698.8 fusion protein. Inone example, covalent associations are between the heterologous sequencecontained in a fusion protein described herein (see, for example, U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in a QUB 698.8-Fc fusionprotein described herein. In another specific example, covalentassociations of fusion proteins described herein are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, for example,oseteoprotegerin (see, for example WO 98/49305). In another embodiment,two or more polypeptides described herein are joined through peptidelinkers. Examples include those peptide linkers described in U.S. Pat.No. 5,073,627. Proteins comprising multiple QUB 698.8 peptides separatedby peptide linkers can be produced using conventional recombinant DNAtechnology.

Multimers may also be prepared by fusing the pro-angiogenic peptides ofthe invention to a leucine zipper or isoleucine zipper polypeptidesequence. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins described herein are those described in WO 94/10308.Recombinant fusion proteins comprising a polypeptide described hereinfused to a polypeptide sequence that dimerizes or trimerizes in solutioncan be expressed in suitable host cells, and the resulting solublemultimeric fusion protein can be recovered from the culture supernatantusing techniques known in the art.

The multimers may also be generated using chemical techniques known inthe art. For example, polypeptides desired to be contained in themultimers described herein may be chemically cross-linked using linkermolecules and linker molecule length optimization techniques known inthe art (see, for example, U.S. Pat. No. 5,478,925). Additionally, themultimers can be generated using techniques known in the art to form oneor more inter-molecule cross-links between the cysteine residues locatedwithin the sequence of the polypeptides desired to be contained in themultimer (see, for example, U.S. Pat. No. 5,478,925). Further,polypeptides described herein may be routinely modified by the additionof cysteine to the N-terminus or C-terminus or biotin to a suitableamino group either at the N-terminus or the side chain of a Lys residueof a peptide of the invention, for example at Lys-4 of SEQ ID NO: 2.Techniques known in the art may be applied to generate multimerscontaining one or more of these modified polypeptides (see, for example,U.S. Pat. No. 5,478,925). Additionally, techniques known in the art canbe used to prepare liposomes containing two or more peptides of theinvention desired to be contained in the multimer (see, for example,U.S. Pat. No. 5,478,925).

Alternatively, those multimers including only naturally-occurring aminoacids can be formed using genetic engineering techniques known in theart. Alternatively, those that include post-translational or othermodifications can be prepared by a combination of recombinant techniquesand chemical modifications. In one embodiment, the pro-angiogenicpeptides of the invention are produced recombinantly using fusionprotein technology described herein or otherwise known in the art (see,for example, U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). For example, polynucleotides coding for ahomodimer described herein can be generated by ligating a polynucleotidesequence encoding a pro-angiogenic peptide of the invention describedherein to sequence encoding a linker polypeptide and then further to asynthetic polynucleotide encoding the translated product of thepolypeptide in the reverse orientation from the original C-terminus tothe N-terminus (lacking the leader sequence) (see, for example, U.S.Pat. No. 5,478,925). The recombinant techniques described herein orotherwise known in the art can be applied to generate recombinantpro-angiogenic peptides of the invention that contain a transmembranedomain (or hydrophobic or signal peptide) and that can be incorporatedby membrane reconstitution techniques into liposomes (see, for example,U.S. Pat. No. 5,478,925).

Pro-Drugs

The peptides described herein are intended, at least in someembodiments, to be administered to a human or other mammal for medicaltreatment. Peptides are typically administered parenterally, and may bereadily metabolized by plasma proteases. Oral administration, which isperhaps the most attractive route of administration, may be even moreproblematic. In the stomach, acid degrades and enzymes break down thepeptides. Those peptides that survive to enter the intestine intact aresubjected to additional proteolysis as they are continuously barraged bya variety of enzymes, including gastric and pancreatic enzymes, exo- andendopeptidases, and brush border peptidases. As a result, passage ofpeptides from the lumen of the intestine into the bloodstream can beseverely limited. However, various prodrugs have been developed thatenable parenteral and oral administration of therapeutic peptides.

Peptides can be conjugated to various moieties, such as polymericmoieties, to modify the physiochemical properties of the peptide drugs,for example, to increase resistance to acidic and enzymatic degradationand to enhance penetration of such drugs across mucosal membranes. Forexample, Abuchowski and Davis have described various methods forderivatizating enzymes to provide water-soluble, non-immunogenic, invivo stabilized products (“Soluble polymers-Enzyme adducts,” Enzymes asDrugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, N.Y.(1981)). Abuchowski and Davis discuss various ways of conjugatingenzymes with polymeric materials, such as dextrans, polyvinylpyrrolidones, glycopeptides, polyethylene glycol and polyamino acids.The resulting conjugated polypeptides retain their biological activitiesand solubility in water for parenteral applications. U.S. Pat. No.4,179,337 teaches coupling peptides to polyethylene glycol orpolypropropylene glycol having a molecular weight of 500 to 20,000Daltons to provide a physiologically active non-immunogenic watersoluble polypeptide composition. The polyethylene glycol orpolypropylene glycol protects the polypeptide from loss of activity andthe composition can be injected into the mammalian circulatory systemwith substantially no immunogenic response.

U.S. Pat. No. 5,681,811, U.S. Pat. No. 5,438,040 and U.S. Pat. No.5,359,030 disclose stabilized, conjugated polypeptide complexesincluding a therapeutic agent coupled to an oligomer that includeslipophilic and hydrophilic moieties. Garmen, et al. describe aprotein-PEG prodrug (Garman, A. J., and Kalindjian, S. B., FEBS Lett.,1987, 223, 361-365). A prodrug can be prepared using this chemistry, byfirst preparing a maleic anhydride reagent from polydispersed MPEG5000and then conjugating this reagent to the peptides disclosed herein. Thereaction of amino acids with maleic anhydrides is well known. Thehydrolysis of the maleyl-amide bond to reform the amine-containing drugis aided by the presence of the neighboring free carboxyl group and thegeometry of attack set up by the double bond. The peptides can bereleased (by hydrolysis of the prodrugs) under physiological conditions.

The peptides can also be coupled to polymers, such as polydispersed PEG,via a degradable linkage, for example, the degradable linkage shown(with respect to pegylated interferon α-2b) in Roberts, M. J., et al.,Adv. Drug Delivery Rev., 2002, 54, 459-476.

The peptides can also be linked to polymers such as PEG using 1, 6 or1,4 benzyl elimination (BE) strategies (see, for example, Lee, S., etal., Bioconjugate Chem., (2001), 12, 163-169; Greenwald, R. B., et al.,U.S. Pat. No. 6,180,095, 2001; Greenwald, R. B., et al., J. Med. Chem.,1999, 42, 3657-3667.); the use of trimethyl lock lactonization (TML)(Greenwald, R. B., et al., J. Med. Chem., 2000, 43, 475-487); thecoupling of PEG carboxylic acid to a hydroxy-terminated carboxylic acidlinker (Roberts, M. J., J. Pharm. Sci., 1998, 87(11), 1440-1445), andPEG prodrugs involving families of MPEG phenyl ethers and MPEGbenzamides linked to an amine-containing drug via an aryl carbamate(Roberts, M. J., et al., Adv. Drug Delivery Rev., 2002, 54, 459-476),including a prodrug structure involving a meta relationship between thecarbamate and the PEG amide or ether (U.S. Pat. No. 6,413,507); andprodrugs involving a reduction mechanism as opposed to a hydrolysismechanism (Zalipsky, S., et al., Bioconjugate Chem., 1999, 10(5),703-707).

Some approaches involve using enzyme inhibitors to slow the rate ofdegradation of proteins and peptides in the gastrointestinal tract;manipulating pH to inactivate local digestive enzymes; using permeationenhancers to improve the absorption of peptides by increasing theirparacellular and transcellular transports; using nanoparticles asparticulate carriers to facilitate intact absorption by the intestinalepithelium, especially, Peyer's patches, and to increase resistance toenzyme degradation; liquid emulsions to protect the drug from chemicaland enzymatic breakdown in the intestinal lumen; and micelleformulations for poorly water-solubulized drugs.

In some cases, the peptides can be provided in a suitable capsule ortablet with an enteric coating, so that the peptide is not released inthe stomach. Alternatively, or additionally, the peptide can be providedas a prodrug. In one embodiment, the peptides are present in these drugdelivery devices as prodrugs.

Free amino, hydroxyl, or carboxylic acid groups of the peptides can beused to convert the peptides into prodrugs. Prodrugs include compoundswherein an amino acid residue, or a polypeptide chain of two or more(e.g., two, three or four) amino acid residues which are covalentlyjoined through peptide bonds to free amino, hydroxy or carboxylic acidgroups of various polymers, for example, polyalkylene glycols such aspolyethylene glycol. Prodrugs also include compounds wherein carbonates,carbamates, amides and alkyl esters are covalently bonded to the abovepeptides through the C-terminal carboxylic acids.

Prodrugs comprising the peptides of the invention or pro-drugs fromwhich peptides of the invention (including analogues and fragments) arereleased or are releasable are considered to be derivatives of theinvention.

Isotopically-labelled peptides or peptide prodrugs are also encompassedby the invention. Such peptides or peptide prodrugs are identical to thepeptides or peptide prodrugs of the invention, but for the fact that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into peptides orpro-drugs of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, and ³⁵S, respectively. Peptides of thepresent invention, prodrugs thereof, and/or the prodrugs which containthe aforementioned isotopes and/or other isotopes of other atoms arewithin the scope of this invention. Certain isotopically-labelledcompounds of the present invention, for example those into whichradioactive isotopes such as ³H and ¹⁴C are incorporated, are useful indrug and/or substrate tissue distribution assays. Tritiated, i.e., ³H,and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for theirease of preparation and detectability. Further, substitution withheavier isotopes such as deuterium, i.e., ²H, can afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements and,hence, may be preferred in some circumstances. Isotopically-labeledpeptides and prodrugs thereof can generally be prepared by carrying outreadily known procedures, including substituting a readily availableisotopically-labeled reagent for a non-isotopically-labeled reagent,e.g., a labeled amino acid.

Peptidomimetics

The present invention further encompasses mimetic peptides of QUB698.8which can be used as therapeutic peptides. Mimetic QUB 698.8 peptidesare short peptides which mimic the biological activity of QUB 698.8.Such mimetic peptides can be obtained from methods known in the art suchas, but not limited to, phage display or combinatorial chemistry. Forexample, the method disclosed by Wrighton, et al., Science 273:458-463(1996) can be used to generate mimetic QUB 698.8 peptides. Thus, in oneembodiment of the invention, the term derivative encompasses suchmimetic QUB 698.8 peptides.

Nucleic Acid

Peptides of and for use in the present invention may be produced by useof nucleic acid in an expression system.

Accordingly the present invention also provides an isolatedpolynucleotide which encodes a peptide of the first aspect of theinvention.

A polynucleotide according to the invention may be provided as anisolate, in isolated and/or purified form, or free or substantially freeof material with which it is naturally associated, such as free orsubstantially free of nucleic acid flanking the gene in the toad genome,except possibly one or more regulatory sequence(s) for expression.Nucleic acid may be wholly or partially synthetic and may includegenomic DNA, cDNA or RNA.

Nucleic acid sequences encoding a peptide in accordance with the presentinvention can be readily prepared by the skilled person using theinformation and references contained herein and techniques known in theart.

Modifications to the sequences can be made, e.g. using site directedmutagenesis, to lead to the expression of modified peptide or to takeaccount of codon preference in the host cells used to express thenucleic acid.

In one embodiment, the nucleic acid comprises the nucleic acid sequence:

-GATTTGCCTAAGATCAAC-. (SEQ ID NO: 10)

However, any polynucleotide which encodes a peptide of the first aspectof the invention is also encompassed by the invention. For example, byvirtue of the degeneracy of the genetic code, polynucleotides, whichencode the QUB698.8 peptide having amino acid sequence SEQ ID NO: 2, butwhich have a nucleic acid sequence which differs from SEQ ID NO: 10 maybe used.

However, for the avoidance of any doubt, in the context of the presentinvention, a polynucleotide which encodes maximakinin, i.e. the aminoacid sequence having Sequence ID No: 1, is not encompassed bypolynucleotides of the present invention.

Polynucleotides of and for use in the present invention may comprise DNAor RNA whose sequences reflect the degeneracy of the genetic code andwhose base composition would reflect nucleic acid type. These may beproduced recombinantly, synthetically, or by any means available tothose in the art, including cloning using standard techniques. Thepolynucleotide may be inserted into any appropriate vector. A furtheraspect of the invention is such a vector comprising a nucleic acid ofthe invention. Any suitable vector may be used in the invention. Forexample, the vector may be a virus (e.g. vaccinia virus, adenovirus,etc.), a plasmid, or cosmid DNA a baculovirus; a yeast vector, a phage,or a chromosome, for example an artificial chromosome.

The nucleic acid may be operably linked to a control sequence which iscapable of providing expression of the nucleic acid in a host cell.Suitable host cells for use in the invention may be prokaryotic oreukaryotic and include bacteria, yeast, insect cells and mammaliancells. Mammalian cell lines which may be used include Chinese hamsterovary cells, baby hamster kidney cells, NSO mouse melanoma cells, monkeyand human cell lines and derivatives thereof and many others.

A host cell strain that modulates the expression of, modifies, and/orspecifically processes the gene product may be used. Such processing mayinvolve glycosylation, ubiquitination, disulfide bond formation andgeneral post-translational modification. Accordingly, the presentinvention also provides a host cell, which comprises one or more nucleicacids or vectors of the invention.

Also encompassed by the invention is a method of producing apro-angiogenic peptide of the invention, the method comprising culturinga host cell comprising a polynuceotide of the invention under conditionsin which expression of the peptide from the polynucleotide occurs and,optionally, isolating and/or purifying the peptide.

For further details relating to known techniques and protocols formanipulation of nucleic acid, for example, in preparation of nucleicacid constructs, mutagenesis, sequencing, introduction of DNA into cellsand gene expression, and analysis of proteins, see, for example, CurrentProtocols in Molecular Biology, 5th ed., Ausubel et al, eds., John Wiley& sons, 2005 and, Molecular Cloning: a Laboratory Manual: 3^(rd) editionSambrook et al., Cold Spring Harbor Laboratory Press, 2001.

Administration

Peptides of and for use in the present invention may be administeredalone but will preferably be administered as a pharmaceuticalcomposition, which will generally comprise a suitable pharmaceuticalexcipient, diluent or carrier selected depending on the intended routeof administration.

The peptides may be administered to a patient in need of treatment viaany suitable route. Some suitable routes of administration include (butare not limited to) oral, rectal, nasal, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural)administration.

For intravenous, injection, or injection at a site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required. Pharmaceuticalcompositions for oral administration may be in tablet, capsule, powderor liquid form. A tablet may comprise a solid carrier such as gelatin oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included.

The composition may also be administered via microspheres, liposomes,other microparticulate delivery systems or sustained releaseformulations placed in certain tissues including blood. Suitableexamples of sustained release carriers include semipermeable polymermatrices in the form of shared articles, e.g. suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,919; EP-A-0058481) copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al,Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) orethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15:167-277, 1981, and Langer, Chem. Tech. 12:98-105, 1982). Liposomescontaining the polypeptides are prepared by well-known methods: DE3,218,121A; Epstein et al, PNAS USA, 82: 3688-3692, 1985; Hwang et al,PNAS USA, 77: 4030-4034, 1980; EP-A-0052522; E-A-0036676; EP-A-0088046;EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Pat. No. 4,485,045 andU.S. Pat. No. 4,544,545. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal rate of the polypeptide leakage.

The peptides and polynucleotides of the invention may be provided in awound dressing. Any suitable wound dressing may be used to deliver suchpeptides or polynucleotides. As will be well known to the skilledperson, many types of wound dressings are conventionally known and maybe used with the peptides or polynucleotides of the invention. These mayinclude but are not limited to hydrocolloid-based wound dressings,hydrogel-based wound dressings, alginate-based wound dressings,foam-based dressings, or anti-microbial dressings.

Hydrocolloid-based dressings generally comprise sodiumcarboxymethylcellulose, gelatine, pectin, elastomers, and adhesivesbonded to a carrier of semi permeable film or a foam sheet to produce aflat, occlusive, adhesive dressing. The dressing forms a gel on thewound surface, thus promoting moist wound healing. Hydrocolloid fibremay be provided as a hydrofibre dressing, from which fibres areconverted from dry dressing to soft coherent gel sheet on contact withexudate.

Hydrogel-based dressing promote wound debridement and consist of amatrix of insoluble polymers with a high water content.

Alginate-based wound dressings comprise calcium alginate, optionallywith sodium alginate. On contact with the wound fluid alginatespartially dissolve to form a hydrophilic gel. They are very suitable forhighly exuding wounds.

Foam dressings may comprise polyurethane or silicone foam.

Anti-microbial dressings may comprise antimicrobial agents, such assilver or iodine containing compounds.

The peptides or polynucleotides of the invention may be used incombination of such dressings and incorporated into such dressings usingtechniques commonly known in the art.

Examples of the techniques and protocols mentioned above and othertechniques and protocols which may be used in accordance with theinvention can be found in Remington: The Science and Practice ofPharmacy, 21st edition, Gennaro A R, et al, eds., Lippincott Williams &Wilkins, 2005.

Targeting therapies may be used to deliver the active agent to a target,e.g. arterial smooth muscle, by the use of targeting systems such asantibodies or cell specific ligands.

Dose

The peptides, polynucleotides and/or compositions of the invention arepreferably administered to an individual in a “therapeutically effectiveamount”, this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage etc, is ultimatelywithin the responsibility and at the discretion of medical physiciansand typically takes account of the disorder to be treated, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners.

Therapeutic Uses

“Treatment” or “therapy” includes any regime that can benefit a human ornon-human animal. The treatment may be in respect of an existingcondition or may be prophylactic (preventative treatment). Treatment mayinclude curative, alleviation or prophylactic effects.

The peptides and methods of the invention may be used in the treatmentof any condition or disorder for which pro-angiogenic molecules and/orvasodilatory molecules may be considered useful. As described above, thepeptides of the invention were found to have potent pro-angiogenicactions. Accordingly, the peptides of the invention may be used to treatany condition for which neovascularization ameliorates or curessymptoms. Such conditions may include coronary artery disease,peripheral vascular disease, ischaemic heart disease, ischaemic diseaseof other organs or organ systems, for example of the peripheral orcentral nervous system, vascular stenoses, occlusion to peripheralvessels of e.g. limbs, and stroke.

The invention may also be used in the treatment of any condition forwhich tissue selective bradykinin receptor agonists may be useful. Asdescribed above, the peptides of the invention were found to have potentvasodilatory actions. Accordingly, the peptides of the invention may beused to treat any condition for which vasodilation ameliorates or curessymptoms. Such conditions may include hypertension, pulmonaryhypertension, coronary artery disease, peripheral vascular disease,ischaemic heart disease, ischaemic disease of other organs or organsystems, vascular stenoses, occlusion to peripheral vessels of e.g.limbs, and stroke. Other conditions for which the invention may findused include the treatment of vasospasm, for example, intra- orpost-operatively, and erectile dysfunction.

In one embodiment the pro-angiogenic effect of the peptides of theinvention may be utilised in the promotion of angiogenesis of collateralvasculature in myocardial tissue during or following ischaemic disease,myocardial infarction or following coronary bypass surgery.

Diseases or conditions which may be treated by the provision of thepeptide(s) of the invention include vascular disease and/or ischaemicdisease causing pathology of the peripheral or central nervous system.Such diseases may include cerebrovascular accidents caused by clotocclusions or by rupture of aneurysms or general/localised ischaemiacausing neuronal death or peripheral functional impairment such as inmotor or sensory functions or speech impairment.

As described herein, the peptide(s) of the invention will be useful intissue repair, for example wound healing, particularly to re-vascularizedamaged tissues or stimulate collateral blood flow during ischemia andwhere new capillary angiogenesis is desired. The peptides may be used inthe repair of any damaged tissue or the healing of any wound wherevascular angiogenesis and/or revascularisation is desired. Thepeptide(s) and methods of the invention may thus be used to treat, forexample, ulcers, such as dermal ulcers, for example pressure sores,venous ulcers, and diabetic ulcers, lacerations, burns, and other tissuetrauma. The peptide(s) and methods may be particularly useful inapplications where transplanted tissues, for example, skin grafts, areemployed in the repair of such wounds.

In vascular graft surgery the peptide(s) and methods of the inventionmay be used to promote endothelialization. Where vascular grafts usingeither transplanted or synthetic material are used the peptide(s) may beapplied to the surface of the graft to promote the growth of vascularsmooth muscle and adventitial cells in conjunction with endothelialcells. Furthermore, where materials, natural or synthetic are to betransplanted into the body, the peptide(s) of the invention may be usedto coat the materials to reduce and/or minimize rejection of thetransplanted material and to stimulate vascularization of thetransplanted materials. Such agonists may also be useful in the repairof vascular damage caused by arteriosclerosis or damage caused byballoon angioplasty. By promoting repair of such tissues, damage may beminimised and restenosis may be prevented.

Alternatively, the peptides may be used for the promotion oracceleration of both internal and external cell growth. In particularthe peptides may be used for the promotion or acceleration of growth ofblood vessels, or dermal cells of the peripheral or central nervoussystem. This may be particularly useful in the promotion of graftrevascularisation, for example in the treatment of burns or aftercosmetic surgery.

The present invention further extends to methods of gene therapy usingnucleic acids encoding peptides of the present invention.

The invention will now be described further in the followingnon-limiting examples. Reference is made to the accompanying drawings inwhich:

FIG. 1 shows the relative molar potencies of the QUB 698.8 peptide onisolated smooth muscle preparations; FIG. 1 a shows the effect of QUB698.8 peptide on rat tail artery and FIG. 1 b shows the effect of theQUB 698.8 peptide on the small intestine.

FIG. 2 a shows the effect of the QUB 698.8 peptide on the migration ofHuman Microvascular Endothelial Cells (HMEC-1) in the wound scrapeassay.

FIG. 2 b shows the effect of the QUB 698.8 peptide at a concentration of1×10⁻⁸M on the time dependent migration of HMEC-1 in the wound assaycompared to a non-treatment control.

FIG. 3 illustrates the effect of the QUB 698.8 peptide on the ability ofHMEC-1 to form tubules on matrigel matrices.

FIG. 4 illustrates the effect of the QUB 698.8 peptide on theproliferation of HMEC-1 over 72 hours compared to a non-treatmentcontrol.

EXAMPLES Materials and Methods

Acquisition of Skin Secretion.

Four adult specimens of Bombina maxima were obtained from a commercialsource and housed in a vivarium under a 12 h/12 h light/dark cycle at22° C. and fed multivitamin-loaded crickets three times per week. Underthese conditions, toads have remained in good health in excess of 3years. Defensive skin secretions were obtained by two methods and underboth sets of conditions, secretions were most pronounced from pairedparatoid and tibial glands. Firstly, the dorsal surface was moistenedwith distilled water followed by three periods of transdermal electricalstimulation (5V, 100 Hz, 140 ms pulse width), each of 10 secondsduration (Tyler et al (1992) J. Pharmacol. Toxicol. Lett. 28, 199-200).Skin secretions were washed from the dorsal skin with distilled water,snap-frozen in liquid nitrogen and lyophilized. The second and preferredtechnique involved gently massaging the dorsal skin surface with alatex-gloved finger that was found to be equally effective in terms ofdry weight secretion yield. Both techniques caused no harm and minimalstress to the animals.

Discovery of QUB 698.8.

QUB 698.8 was identified in reverse phase HPLC fractions of maximakininthat had been incubated with human saliva for 2 hours. It was generatedfrom its parent peptide, maximakinin, by combined sequential proteolysisby the cocktail of proteases present in saliva.

Smooth Muscle Bioassays

(A) Arterial Smooth Muscle:

Male albino Wistar rats (200-350 g) were euthanized by asphyxiationfollowed by cervical dislocation.

The tail artery was prepared as previously described (Hirst et al (1994)Br. J. Radiol. 67, 795-799). Incubation buffer was 95% O₂/5% CO₂oxygenated Krebs' solution (NaCl 118 mM, KCl 4.7 mM, NaHCO₃ 25 mM, NaH₂PO₄ 1.15 mM, CaCl₂ 2.5 mM, MgCl₂ 1.1 mM, glucose 5.6 mM). Constrictionor dilation of the arterial smooth muscle preparation was detected by anincrease or decrease in pressure generated by water column displacementusing pressure transducers connected to a MacLab System (AD InstrumentsPty Ltd. Australia). Data were displayed graphically on a Macintoshcomputer. Viability was determined using a range of bolus phenylephrine(5 μM-10 μM) exposures and the endothelial layer of the artery wasremoved by bubbling with oxygen for 10 s. Absence of the endotheliallayer was confirmed by the lack of relaxation in response to a 30 minperfusion of acetylcholine (50 μM) after preconstriction withphenylephrine (10 μM).

(B) Small Intestinal Smooth Muscle:

For intestinal smooth muscle preparations, one cm thick rings of ileumwere carefully placed onto the pins of a MacLab force transducer, onepin acting as a stationary fixed point while the second pin was free,permitting application of tension to the smooth muscle. The muscle ringswere gradually exposed to 0.1 g increments in resting tension until thespontaneous contractions originated from a resting tension of 0.5 g. Thecontracting muscle preparations were allowed to stabilise for 25 minbefore the application of peptides. After perfusion of arterialpreparations with 10 μM phenylephrine to obtain constriction plateaux,relative relaxation was recorded following applications of peptides inthe range of 1×10⁻⁵-1×10⁻¹⁰ M. The intestinal smooth muscle ringpreparations were exposed to peptide doses as above and relative changesin tension were recorded.

Results

-   -   1. Application of QUB 698.8 to a rat tail arterial smooth muscle        preparation produced a dose-dependent relaxation. Detailed        responses compared to bradykinin and maximakinin are summarized        in Table 1—panel A and FIG. 1 a. QUB 698.8 was almost equipotent        with bradykinin in this bioassay but more potent than        maximakinin.    -   2. Application of QUB 698.8 to a rat ileal smooth muscle ring        preparation produced a dose-dependent constriction. Detailed        responses compared to bradykinin and maximakinin are summarized        in Table 1—panel A and FIG. 1 b. QUB 698.8 was less potent than        both bradykinin and maximakinin in this bioassay. These combined        data show an optimum concentration of QUB 698.8 two orders of        magnitude lower for induction of maximal effect in arterial        compared to ileal smooth muscle.    -   3. Selective arterial smooth muscle dilators often have        pro-angiogenic properties so QUB 698.8 was subjected to a range        of appropriate bioassays to determine spectra of activity.    -   4. In the human microvessel endothelial cell wound scrape assay,        cells are grown on a glass slide to confluence, a standard-sized        wound is made with a pipette tip and time taken to repair tear        is measured. QUB 698.8 promoted wound closure 42% faster than        non-treated controls at an optimum effective concentration of 10        nanomolar (Table 1—panel B and FIGS. 2 a and b).    -   5. Using an in vitro endothelial cell angiogenic tubule        formation assay (Matrigel) QUB 698.8 induced 75% more tubules        than time-matched controls at an optimum concentration of 1        nanomolar (Table 1—panel B and FIG. 3).    -   6. Using a standard MTT assay to monitor potential cell        proliferative effects, QUB 698.8 caused a 70% increase in        endothelial cell number after 24 h when compared to non-treated        controls although there were no significant differences after        48 h. (Table 1—panel B and FIG. 4). This may be indicative of a        rapid role in cell cycle synchronization.

Taken together, these data are indicative of a potent role for QUB 698.8in promoting the growth of new blood vessels at concentrations thatwould render this peptide one of the most potent agents known atpresent. The results also indicate potency in effecting the growth ofother dermal cell types in accelerating wound closure.

QUB 698.8, fragments and derivatives thereof will thus have clinicalefficacy in promoting angiogenesis and in wound and tissue repair ofboth surface and internal tissues.

The mechanism of action by which the peptides of the invention exertsits angiogenic effects may be through binding a receptor or may involvea binding site that is not in the truest sense a receptor.

Without being bound by any one particular theory, it is believed that,due to its total structural dissimilarity to bradykinin receptorligands, the target for the peptide is not likely to be a bradykininreceptor. It does not apparently work directly through cytoskeletalmodifications. This distinguishes its pro-angiogenic effects fromC-terminal fragments of maximakinin that do act through bradykininreceptors.

Furthermore, the potent selective vasodilatory effects herein supportthe peptide's use in applications in which vasodilation is desired.

All documents referred to in this specification are herein incorporatedby reference. Various modifications and variations to the describedembodiments of the inventions will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes of carrying out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention.

TABLE 1A Physiological Screens QUB698.8 EC₅₀ % Maximum Optimum Mx Ec₅₀Name of Screen Peptide Activity Response Concentration Bk EC₅₀ Rat TailArtery Relaxant of pre- 25% relaxation 10⁻⁷ M 7.00 × 10⁻⁹ M constricted30% relaxation 10⁻⁷ M 6.18 × 10⁻⁹ M artery 32% relaxation 10⁻⁶ M 1.18 ×10⁻⁸ M Rat Ileum Constriction of 52% Constriction 10⁻⁵ M 1.44 × 10⁻⁶ Mspontaneously 52% Constriction 10⁻⁶ M 5.34 × 10⁻⁷ M contracting 37%Constriction 10⁻⁶ M 1.18 × 10⁻⁸ M ileum

TABLE 1B In vitro angiogenic screens Name of Peptide Optimum Maximum %Other screen effect Concentration EC₅₀ Effect information MigrationPotent 10⁻⁸ M 3.39 × 10⁻¹⁰ 42% Increase in 50% wound wound promoter ofwound closure closure in scrape T = 7 wound compared to T = 3 hrs doseclosure time matched response control Wound Potent 10⁻⁸ M — — Completescrape total stimulator of wound time to initial closure in 16 closureendothelial hrs migration Endothelial Potent 10⁻⁹ M 5.26 × 10⁻¹¹ 75%increase in cell tubule stimulator of tubule formation formationendothelial compared to assay tubule time matched formation controlEndothelial Potent 10⁻⁸ M 6.59 × 10⁻¹¹ 70% increase in No further cellstimulator of cell number increase in proliferation endothelial after 24hrs proliferation assay cell observed proliferation after 24 hrs

1. The peptide consisting of the amino acid sequence:Asp-Leu-Pro-Lys-Ile-Asn. (SEQ ID NO: 2)


2. A pharmaceutical composition comprising the peptide according toclaim 1 and a pharmaceutically acceptable carrier or excipient.
 3. Acomposition combination comprising a wound dressing containing and thepeptide according to claim 1.